Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: impact of his-tagging and carotenoid type

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCPs studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and the time scales on which these modifications affect photoactivation. The presence of a His-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP. - Complete functional characterization of Synechocystis and Planktothrix OCPs - Hitherto unknown structures of ECN- and CAN-functionalized Planktothrix OC

ENZO: A Web Tool for Derivation and Evaluation of Kinetic Models of Enzyme Catalyzed Reactions

We describe a web tool ENZO (Enzyme Kinetics), a graphical interface for building kinetic models of enzyme catalyzed reactions. ENZO automatically generates the corresponding differential equations from a stipulated enzyme reaction scheme. These differential equations are processed by a numerical solver and a regression algorithm which fits the coefficients of differential equations to experimentally observed time course curves. ENZO allows rapid evaluation of rival reaction schemes and can be used for routine tests in enzyme kinetics. It is freely available as a web tool, at http://enzo.cmm.ki.si

Intravesicle Isothermal DNA Replication

<p>Abstract</p> <p>Background</p> <p>Bacterial and viral DNA replication was previously reconstituted <it>in vitro </it>from component parts <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr><abbr bid="B3">3</abbr><abbr bid="B4">4</abbr></abbrgrp>. Significant advances in building minimal cell-like structures also have been made recently <abbrgrp><abbr bid="B5">5</abbr><abbr bid="B6">6</abbr><abbr bid="B7">7</abbr></abbrgrp>. Combining the two approaches would further attempts to build a minimal cell-like structure capable of undergoing evolution by combining membrane encapsulation and genome replication. Towards this end, we attempted to use purified genomic replication protein components from thermophilic bacterial sources to copy strands of DNA isothermally within lipid vesicles.</p> <p>Findings</p> <p>Bacterial replication components (such as helicases and DNA polymerases) are compatible with methods for the generation of lipid vesicles. Encapsulation inside phospholipid vesicles does not inhibit the activity of bacterial DNA genome replication machinery. Further the described system is efficient at isothermally amplifying short segments of DNA within phospholipid vesicles.</p> <p>Conclusions</p> <p>Herein we show that bacterial isothermal DNA replication machinery is functional inside of phospholipid vesicles, suggesting that replicating cellular mimics can be built from purified bacterial components.</p

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern whether this experimental approach allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments such that biologically relevant insight emerges

Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes

Molecular fingerprinting of radiation resistant tumors: Can we apprehend and rehabilitate the suspects?

Radiation therapy continues to be one of the more popular treatment options for localized prostate cancer. One major obstacle to radiation therapy is that there is a limit to the amount of radiation that can be safely delivered to the target organ. Emerging evidence suggests that therapeutic agents targeting specific molecules might be combined with radiation therapy for more effective treatment of tumors. Recent studies suggest that modulation of these molecules by a variety of mechanisms (e.g., gene therapy, antisense oligonucleotides, small interfering RNA) may enhance the efficacy of radiation therapy by modifying the activity of key cell proliferation and survival pathways such as those controlled by Bcl-2, p53, Akt/PTEN and cyclooxygenase-2. In this article, we summarize the findings of recent investigations of radiosensitizing agents in the treatment of prostate cancer

β-hairpin-mediated formation of structurally distinct multimers of neurotoxic prion peptides

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A117V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies

Cancers of unknown primary origin: current perspectives and future therapeutic strategies

It is widely accepted that systemic neoplastic spread is a late event in tumour progression. However, sometimes, rapidly invasive cancers are diagnosed because of appearance of metastatic lesions in absence of a clearly detectable primary mass. This kind of disease is referred to as cancer of unknown primary (CUP) origin and accounts for 3-5% of all cancer diagnosis. There is poor consensus on the extent of diagnostic and pathologic evaluations required for these enigmatic cases which still lack effective treatment. Although technology to predict the primary tumour site of origin is improving rapidly, the key issue is concerning the biology which drives early occult metastatic spreading. This review provides the state of the art about clinical and therapeutic management of this malignant syndrome; main interest is addressed to the most recent improvements in CUP molecular biology and pathology, which will lead to successful tailored therapeutic options

ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules.

Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics. Recent studies have demonstrated the capability of ESI-IMS-MS to identify small molecule modulators of amyloid assembly and to determine the mechanism by which they interact (positive, negative, non-specific binding, or colloidal) in a high-throughput format. Here, we demonstrate these advances using self-assembly of Aβ40 as an example, and reveal two new inhibitors of Aβ40 fibrillation

Structure-Based Design of Functional Amyloid Materials

Amyloid fibers, once exclusively associated with disease, are acquiring utility as a class of biological nanomaterials. Here we introduce a method that utilizes the atomic structures of amyloid peptides, to design materials with versatile applications. As a model application, we designed amyloid fibers capable of capturing carbon dioxide from flue gas, to address the global problem of excess anthropogenic carbon dioxide. By measuring dynamic separation of carbon dioxide from nitrogen, we show that fibers with designed amino acid sequences double the carbon dioxide binding capacity of the previously reported fiber formed by VQIVYK from Tau protein. In a second application, we designed fibers that facilitate retroviral gene transfer. By measuring lentiviral transduction, we show that designed fibers exceed the efficiency of polybrene, a commonly used enhancer of transduction. The same procedures can be adapted to the design of countless other amyloid materials with a variety of properties and uses